Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
The flower and ornamental plant industry strives to develop new and different varieties of flowers and/or plants. In the flower industry in particular, an effective way to create such novel varieties is through the manipulation of flower colour where classical breeding techniques have been used with some success to produce a wide range of colours for most of the commercial varieties of flowers and/or plants. This approach has been limited, however, by the constraints of a particular species' gene pool and for this reason it is rare for a single species to have the full spectrum of coloured varieties. For example, the development of novel coloured varieties of plants or plant parts such as flowers, foliage and stems would offer a significant opportunity in both the cut flower and ornamental markets.
In the flower industry, the development of novel coloured varieties of major species such as rose, chrysanthemum, tulip, lily, carnation, gerbera, orchid, lisianthus, begonia, torenia, geranium, petunia and nierembergia would be of great interest. A more specific example would be the development of a blue rose or gerbera for the cut flower market.
In addition, the development of novel coloured varieties of plant parts such as vegetables, fruits and seeds would offer significant opportunities in agriculture, for example, novel coloured seeds would be useful as proprietary tags for plants.
Flower and fruit colour is predominantly due to flavonoids which contribute a range of colours from yellow to red to blue. The flavonoid molecules which make the major contribution to flower colour are the anthocyanins which are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and are localized in the vacuole.
The flavonoid pigments are secondary metabolites of the phenylpropanoid pathway. The biosynthetic pathway for the flavonoid pigments (flavonoid pathway) is well established, (Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979; Wiering and De Vlaming, 1984; Schram et al., 1984; Stafford, 1990, Holton and Cornish, 1995) and is shown in FIGS. 1A and B. Three reactions and enzymes are involved in the conversion of phenylalanine to p-coumaroyl-CoA, one of the first key substrates in the flavonoid pathway. The enzymes are phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate: CoA ligase (4CL). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA (provided by the action of acetyl CoA carboxylase (ACC) on acetyl CoA and CO2) with one molecule of p-coumaroyl-CoA. This reaction is catalyzed by the enzyme chalcone synthase (CHS). The product of this reaction, 2′,4,4′,6′, tetrahydroxy chalcone, is normally rapidly isomerized by the enzyme chalcone flavanone isomerase (CHI) to produce naringenin. Naringenin is subsequently hydroxylated at the 3 position of the central ring by flavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK).
The B-ring of DHK can be hydroxylated at either the 3′, or both the 3′ and 5′ positions, to produce dihydroquercetin (DHQ) and dihydromyricetin (DHM), respectively. The pattern of hydroxylation of the B-ring plays a key role in determining petal colour, with DHK generally leading to the production of the brick red pelargonidin-based pigments, DHQ generally leading to the red/pink cyanidin-based pigments and DHM generally leading to the blue/violet delphinidin-based pigments.
The dihydroflavonols (DHK, DHQ and DHM) can also be acted upon by flavonol synthase to produce the flavonols kaempferol, quercetin and myricetin. The flavonols are colourless but act as copigments with the anthocyanins to enhance flower colour.
The next step in the pathway, leading to the production of the coloured anthocyanins from the dihydroflavonols, involves dihydroflavonol-4-reductase (DFR) with the production of the leucoanthocyanidins. These flavonoid molecules are unstable under normal physiological conditions and glycosylation at the 3-position, through the action of glycosyltransferases, stabilizes the anthocyanidin molecule thus allowing accumulation of the anthocyanins. In general, the glycosyltransferases transfer the sugar moieties from UDP sugars and show high specificities for the position of glycosylation and relatively low specificities for the acceptor substrates (Seitz and Hinderer, 1988). Anthocyanins can occur as 3-monosides, 3-biosides and 3-triosides as well as 3,5-diglycosides and 3,7-diglycosides associated with the sugars glucose, galactose, rhamnose, arabinose and xylose (Strack and Wray, 1993).
Glycosyltransferases involved in the stabilization of the anthocyanidin molecule include UDP glucose: flavonoid 3-glucosyltransferase (3GT), which transfers a glucose moiety from UDP glucose to the 3-O-position of the anthocyanidin molecule to produce anthocyanidin 3-O-glucoside. In petunia and pansy (amongst others), these anthocyanins can then be glycosylated by another glycosyltransferase, UDP rhamnose: anthocyanidin 3-glucoside rhamnosyltransferase (3RT), which adds a rhamnose group to the 3-O-bound glucose of the anthocyanin molecule to produce the anthocyanidin 3-rutinosides, and once acylated, can be further modified by UDP glucose: anthocyanidin 3-(p-coumaroyl)-rutinoside 5 glucosyltransferase (5GT).
Many anthocyanidin glycosides exist in the form of polyacylated derivatives. Acylation may be important for uptake of anthocyanins into the vacuoles as was demonstrated by Hopp and Seitz (1987). The acyl groups that modify the anthocyanidin glycosides can be divided into 2 major classes based upon their structure. The aliphatic acyl groups such as malonic acid or succinic acid and the aromatic class such as the hydroxy cinnamic acids including p-coumaric acid, caffeic acid and ferulic acid and the benzoic acids such as p-hydroxybenzoic acid. Aromatic acyl groups have been reported to cause intra and/or intermolecular co-pigmentation that leads to the stabilization of the anthocyanin molecule with a bathochromic shift (a positive shift in the wavelength of the maximum of absorption of the visible band) and a subsequent bluing of the colour (Dangles et al., 1993: Lu et al., 1992) (Brouillard and Dangles, 1993). In fact many blue flowers have been shown to contain aromatically acylated delphinidin pigments (Goto and Kondo, 1991).
A number of plants contain anthocyanins aromatically acylated at a glucose (Brouillard and Dangles, 1993) that may be attached to the anthocyanin molecule at positions C3, C5, C7, C3′ or C5′ (see Strack and Wray (1993) for figure of anthocyanin structure). For example, Perilla ocimoides has been shown to contain the anthocyanin shisonin in which coumaric acid is bound to glucose at position C3 of cyanidin 3,5-diglucoside (Goto et al., 1987). Gentiana makinoi has been shown to contain the anthocyanin gentiodelphin which contains an aromatic acyl group attached to a glucose at position C3′ and an aromatic acyl group attached to a glucose at position C5 (Yoshida et al., 1992). However, the anthocyanins in Petunia hybrida flowers are acylated by p-coumaric acid or caffeic acid at a rhamnose group attached to a glucose group at position C3 to produce anthocyanidin 3-p-coumaroylrutinoside 5-glucosides and anthocyanidin 3-caffeoylrutinoside 5-glucosides (Griesbach et al., 1991). This is also the case in a number of other flowers such as petals of Silene dioica (Kamsteeg et al., 1980), flowers of Viola tricolour contain violanin (delphinidin 3-coumaroylrutinoside 5-glucoside) (Goto et al., 1978), Lobelia erinus flowers contain anthocyanins with a 3-coumaroylrutinoside group (Kondo et al., 1989), Iris ensenta flowers contain malvidin 3-coumaroylrutinoside-5-glucoside, petunidin 3-coumaroylrutinoside-5-glucoside and delphinidin 3-coumaroylrutinoside-5-glucoside (Yabuya, T., 1991) and Eustoma grandiflorum flowers contain delphinidin 3-coumaroylrhamnosylgalactoside-5-glucoside and pelargonidin 3-coumaroylrhamnosylgalactoside-5-glucoside (Asen et al., 1986). All of which would probably produce an aromatic acyltransferase that is able to attach an aromatic acyl group to the rhamnose group that is attached to a glycosyl at position C3 of the anthocyanin molecule.
The isolation of flavonoid aromatic acyltransferases which transfer aromatic acyl groups to the glucose attached to the flavonoid molecule has been disclosed in PCT/JP96/00348 (International Patent Publication No. WO 96/25500) entitled A gene encoding a protein having acyl group transfer activity. These sequences include the 5-aromatic acyltransferase from Gentiana triflora (Fujiwara et al., 1998), the encoded amino acid sequences of anthocyanidin-glucoside aromatic acyltransferases from Gentiana triflora (pGAT4 and pGAT106), Senecio cruentus (pCAT8), Lavandula angustifolia (pLAT21), Perilla ocimoides (pSAT8) and a Petunia hybrida homologue (pPAT48).
In addition to the above modifications, pH and copigmentation with other flavonoids such as flavonols and flavones can affect petal colour. Flavonols and flavones can also be aromatically acylated (Brouillard and Dangles, 1993).
The ability to control the activity of anthocyanidin 3-rutinoside acyltransferase would provide a means of manipulating petal colour thereby enabling a single species to express a broader spectrum of flower colours. Such control may be by modulating the level of production of an indigenous enzyme or by introducing a non-indigenous enzyme.